1. Field of the Invention
This invention is directed to methods for the preparation of monofucosylated and sialylated derivatives of the compound .beta.Gal(1-4).beta.GlcNAc-(1-3).beta.Gal(1-4).beta.GlcNAc-OR. In particular, the methods of this invention provide for a multi-step synthesis wherein selective monofucosylation is accomplished on the 3-hydroxy group on only one of the GlcNAc units found in the .beta.Gal(1-4).beta.GlcNAc-(1-3).beta.Gal(1-4).beta.GlcNAc-OR compound. In these methods, monofucosylation is achieved by the use of an .alpha.(1-3)fucosyltransferase. This invention is also directed to compounds prepared by the herein described methods.
2. References
The following references are cited in this application as superscript numbers at the relevant portion of the application:
1a. Feizi, TIBS, 16:84-86 (1991) PA0 1b. Springer et al., Nature, 349:196-197 (1991) PA0 1c. McEver et al., Thromobosis and Haemostasis, 66:80-87 (1991) PA0 2. Sabesan et al., J. Amer. Chem. Soc., 108:2068-2080 (1986) PA0 3. Toone et al., Tetrahedron 45:5365-5422 (1989) PA0 4. Palcic et al., Carbohydr. Res., 190:1-11 (1989) PA0 5. Walz et al., Science, 250:1132-1135 (1990) PA0 6. Phillips et al., Science, 250:1130-1132 (1990) PA0 7. Tiemeyer et al., Proc. Natl. Acad. Sci. USA, 88:1138-1142 (1991) PA0 8. Holmes et al., J. Biol. Chem., 261:3737-3743 (1986) PA0 8b. Holmes et al., Arch. Biochem. Biophys., 274:633-647 (1989) PA0 8c. Basu et al., Indian J. Biochem. Biophys., 25:112-118 (1988) PA0 8d. Hanisch et al., 178:23-28 (1988) PA0 9. Fukuda et al., J. Biol. Chem., 261:2376-2383 (1986) PA0 10. Nudelman et al., J. Biol. Chem., 263:13942-13951 (1988) PA0 11. Howard et al., J. Biol. Chem., 262:16830-16837 (1987) PA0 12. Johnson et al., Biochem. Soc. Trans. p.396 (1987) PA0 13. Foster et al., J. Biol. Chem., 266:3526-3531 (1991) PA0 14. Smith et al., J. Biol. Chem., 262:12040-12047 (1987) PA0 15. Paulson et al., J. Biol. Chem., 253:5617-5624 (1978) PA0 16. Ichikawa et al., J. Amer. Chem. Soc. 113:4698-4700 (1991) PA0 17. Nilsson et al., J. Carbohydr. Chem., 9:1-19 (1990) PA0 18. Gokhale et al., Can. J. Chem., 68:1063-1071 (1990) PA0 19. Mazid et al., U.S. Pat. No. 5,059,535, Oct. 22, 1991. PA0 20. Weinstein et al., J. Biol. Chem., 257:13835-13844 (1982) PA0 21. Unverzagt et al., J. Amer. Chem. Soc., 112:9308-9309 (1990) PA0 22. Ippolito et al., U.S. patent application Ser. No. 07/714,161 filed Jun. 10, 1991 PA0 23. Sialic Acids in "Cell Biology Monographs" Schauer, Editor, 10:6 (1982) PA0 24. Reuter et al., Glycoconjugate J., 5:133-135 (1988) PA0 25. Weinstein et al., J. Biol. Chem., 257:13835-13844 (1982) PA0 26. Paulson et al., J. Biol. Chem., 252:2356-2362 (1977) PA0 27. Paulson, Agnew. Chem. Int. Ed. Eng., 21:155-173 (1982) PA0 28. Schmidt, Agnew. Chem. Int. Ed. Eng., 25:212-235 (1986) PA0 29. Fugedi et al., Glycoconj. J., 4:97-108 (1987) PA0 30. Alais et al., Carbohydr. Res., 207:11-31 (1990) PA0 31. Smith and Ziola, Immunology, 58:245 (1986) PA0 32. Sleytr et al., Arch. Microbiol., 146:19 (1986) PA0 33. Lowe et al., Cell, 63:475-485 (1990) PA0 34. Macher et al. Glycobiology 1(6):577-584 (1991) PA0 35. Lowe et al., J. Biol. Chem., 266:17467-17477 (1991) PA0 36. Piller et al., J. Biol. Chem., 258:12293-12299 (1983) PA0 37. Hosimi et al., Japan J. Med. Sci. Biol. 42:77-82 (1989) PA0 38. Yates et al., Carbohydr. Res., 120:251-268 (1983) PA0 39. Hosomi et al., J. Biochem., 95:1655-1659 (1987) PA0 40. Zielenski et al., FEBS Lett. 158:164-168 (1983) PA0 41. Hosimi et al., Japan J. Med. Sci. Biol. 38:1-8 (1985) PA0 42. Van den Eijnden et al., J. Biol. Chem., 258: 3435-3437 (1983) PA0 43. Van den Eijnden et al., J. Biol. Chem., 263:12461-12471 (1988) PA0 44. Basu et al., J. Biol. Chem., 259:12557-12562 (1984) PA0 45. Hosomi et al., Jpn. S. Vet. Sci. 51:1-6 (1989) PA0 46. Holmes et al., J. Biol. Chem. 262:15649-15658 (1987) PA0 47. Palcic et al., Glycobiology, 1:205-209 (1991) PA0 48. Palcic et al., Carbohydr. Res., 159:315-324 (1987) PA0 49. Wong et al., J. Am. Chem. Soc., 113:8137-8145 (1991) PA0 50. Ekberg et al., Carbohydr. Res. 110:55-67 (1982) PA0 51. Dahmen et al., Carbohydr. Res. 118:292-301 (1983) PA0 52. Rana et al., Carbohydr. Res. 91:149-157 (1981) PA0 53. Amvam-Zollo et al., Carbohydr. Res. 150:199-212 (1986) PA0 54. Paulsen et al., Carbohydr. Res. 104: 195-219 (1982) PA0 55. Chernyak et al., Carbohydr. Res. 128:269-282 (1984) PA0 56. Fernandez-Santana et al., J. Carbohydr. Chem. 8:531-537 (1989) PA0 57. Lee et al., Carbohydr. Res., 37:193 et seq. (1974) PA0 58. Ratcliffe et al., U.S. patent application Ser. No. 07/278,106, filed Nov. 30, 1988 PA0 59. Jiang et al., "Chemical Synthesis of GDP-Fucose", U.S. patent application Ser. No. 07/848,223 filed Mar. 9, 1992 PA0 60. Weinstein et al., J. Biol. Chem., Vol. 257, No. 22, pp. 13835-13844 (9182) PA0 61. Lemieux et al., Can. J. Chem., 58:631-653 (1980) PA0 62. Ichikawa et al., Anal. Biochem., 202:215-238 (1992) PA0 63. Schenkman et al., Cell, 65:1117-1125 (1991) PA0 64. Thiem et al., Angew. Chem. Int. Ed., 30(11):1503-1505 (1991) PA0 65. Venot et al., U.S. patent application Ser. No. 07/771,259, filed Oct. 2, 1991 PA0 66. Lemieux et al., U.S. Pat. No. 4,137,401, Jan. 30, 1979 PA0 67. Matsumoto et al., Anal. Biochem., 116 (1981) 103-110 PA0 68. Kukowska-Latallo et al., Genes and Development, 4:1288-1303 (1990) PA0 69. Dumas et al., Bioorg. Med. Letters, 1:425-428 (1991) PA0 70. Prieels et al., J. Biol. Chem., 256:10456-10463 (1981) PA0 71. Eppenberger-Castori et al., Glycoconj. J. 6:101-114 (1989) PA0 72. Nunez, et al., Can. J. Chem., 59:2086-2095 (1981) PA0 73. Gokhale et al., Can. J. Chem., 68:1063-1071 (1990) PA0 74. Schmidt, et al., Liebigs Ann. Chem., 121-124 (1991) PA0 75. Veeneman, et al., Tetrahedron Lett., 32:6175-6178 (1991)
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
3. State of the Art
The art teaches that specific oligosaccharides such as sialylated and fucosylated structures are involved as ligands in cell adhesion phenomena..sup.1a-1c Similarly, oligosaccharide glycosides relating to blood group determinant structures have been found to impart immunosuppressive and tolerogenic properties to mammals when the mammals were previously challenged with an antigen. See Ippolito et al..sup.22, which application is incorporated herein by reference in its entirety. In this regard, oligosaccharide glycosides relating to blood group determinant structures include the compound .beta.Gal(1-4).beta.GlcNAc(1-3).beta.Gal(1-4).beta.GlcNAc-OR depicted in FIG. 1 of this application as compound 1a.
Ippolito et al..sup.22 further discloses that blood group determinant oligosaccharide glycosides having a sialic acid group (or an analogue thereof) at the non-reducing sugar terminus of the oligosaccharide glycosides and which are also monofucosylated possess immunosuppressive and tolerogenic properties (e.g., sialyl Lewis.sup.X --Compound III in FIG. 12 of Ippolito et al..sup.22).
In view of the above, we desired to prepare a monofucosylated derivative of compound 1a having a sialic acid group (or an analogue thereof) at the non-reducing sugar terminus of this compound wherein the fucosyl group was pendant to the 3-hydroxy of only one of the GlcNAc groups.
A synthetic approach employing enzymatic sialylation and fucosylation steps is particularly appropriate in order to provide an efficient route for the preparation of sialylated and monofucosylated derivatives of compound 1a. In this regard, since the work of Sabesan et al.,.sup.2 -sialyltransferases, mostly the .beta.Gal(1-4).beta.GlcNAc .alpha.(2-6)- and the .beta.Gal(13/4).beta.GlcNAc .alpha.(2-3)-sialyltransferases from rat liver and the .beta.Gal(1-3).beta.GalNAc .alpha.(2-3)sialyltransferase from porcine submaxillary gland have often been used for synthetic purposes..sup.3 The former two sialyltransferases are useful in sialylating a terminal .beta.Gal(1-4).beta.GlcNAc- group in an oligosaccharide glycoside. The latter sialyltransferase which has a wide acceptor specificity, is useful in sialylating a terminal .beta.Gal(1-3).beta.GlcNAc-group in oligosaccharide glycosides based on the Lewis.sup.c (Type I) backbone.sup.4 but, because of the low affinity of this enzyme for the Type II backbone, the synthesis of sialylated N-acetyllactosaminyl structures, such as those present in the sialyl Lewis.sup.x,.sup.5 sialyl dimeric Lewis.sup.x6 or the corresponding internally monofucosylated derivative.sup. 7, by use of this sialyltransferase is much more difficult..sup.4
By using fucosyltransferases of various specificities, the biosynthetic pathway leading to sialyl Lewis.sup.x and the sialylated dimeric Lewis.sup.x structures has been shown to proceed by the sequential sialylation followed by fucosylation of the Type II precursors..sup.8a-d A similar process "extension, sialylation, fucosylation" has also been proposed.sup.9 to lead to internally fucosylated repetitive Type II terminal structures, such as: .alpha.Neu5Ac(2-3).beta.Gal(1-4).beta.GlcNAc(1-3).beta.Gal(1-4)-[ .alpha.Fuc(1-3)].beta.GlcNAc-..sup.9 The identification of the new terminal structure .beta.Gal(1-4).beta.GlcNAc(1-3).beta.Gal(1-4)-[[.alpha.Fuc(1-3)].beta.GlcN Ac-, defined by the antibody ACFH-18.sup.10, led to a proposed new biosynthetic pathway such as "elongation followed by selective internal fucosylation". While patterns of initial internal monofucosylation of di-N-acetyllactosaminyl glycolipids have been observed for fucosyltransferases present in LECII Chinese Hamster Ovary mutant.sup.11 and in human colonic adenocarcinoma Colo 205 cells.sup.8b, these fucosyltransferases are not readily available and/or do not selectively lead to monofucosylated structures. Similarly, while fucosyltransferases possessing the specificity required for the synthesis of the internally fucosylated structure .alpha.Neu5Ac(2-3).beta.Gal(1-4)GlcNAc-.beta.Gal(1-4)[.alpha.Fuc(1-3)]GlcN Ac have been identified.sup.34 and in one case a recombinant enzyme.sup.35 has been identified, their availability is also limited. Moreover, other .alpha.(1-3)fucosyltransferases do not transfer L-fucose onto N-acetylglucosamine moieties found in acceptors possessing a terminal .alpha.Neu5Ac(2-3).beta.Gal(1-4 ).beta.GlcNAc- sequence..sup.12,13,34 It has already been noted that "the order of addition of .alpha.(1-3) fucose in N-acetyllactosaminyl sequences of glycoconjugates will then depend upon the particular .alpha.(1-3)fucosyltransferase present".sup.11.
The single fucosylation at the internal N-acetylglucosamine unit of the .alpha.(2-6)sialyl di-N-acetyllactosaminyl sequence leading to the terminal structure .alpha.Neu5Ac(2-6).beta.Gal(1-4).beta.GlcNAc(1-3)-Gal(1-4)-[.alpha.Fuc(1-3 )].beta.GlcNAc-.sup.8a (also proposed for a sialylfucopentaose from human milk.sup.14) is in agreement with the proposed mutually exclusive glycosylation pattern of the .beta.Gal(1-4).beta.GlcNAc .alpha.(2-6)sialyltransferase and the .beta.Gal(13/4).beta.GlcNAc .alpha.(13/4)fucosyltransferase in the synthesis of asparaginyl linked oligosaccharides in glycoproteins..sup.15
In view of the above, processes which would enzymatically prepare sialylated and monofucosylated derivatives of compound 1a without the need to employ a fucosyltransferase specific for monofucosylation would be particularly desirable.
The present invention is based, in part, on the discovery of synthetic pathways which utilizes enzymatic fucosylation and sialylation steps and which result in the selective formation of monofucosylated derivatives of compound 1a without the need to employ a fucosyltransferase which is specific for monofucosylation on either of the GlcNAc units of compound 1a.